There are over two hundred documented diseases of cultivated tomato (Compendium of Tomato Diseases. J. B. Jones, J. P. Jones, R. E. Stall, T. A. Zitter, editors, (1997) American Phyto-pathological Society Press, St. Paul, Minn.). To combat the damage caused by these pathogens, growers typically employ an integrated pest management strategy including both cultural practices and pesticide use. An example of a cultural practice is the use of netting over tomato plants, which provides a physical barrier that can be effective in excluding disease-bearing insects from infecting the crop.
Despite numerous research studies that have demonstrated efficacious transgenic approaches against plant diseases, there are currently no transgenic tomato varieties available to the grower that are resistant to any pathogens. Further, there remains an issue of public resistance, particularly in the European Union, which, combined with the high cost of obtaining regulatory approval, have effectively prohibited this promising technology from being used in commercial tomato cultivation.
Introgression of disease resistance genes into modern cultivars using traditional breeding approaches has remained an effective technology available for combating the majority of plant diseases. Because of its continued success, the approach is still a primary focus in both academic and commercial tomato breeding programs.
Among the hundreds of tomato pathogens, diseases caused by nematodes and the Tomato yellow leaf curl virus (TYLCV) are among the most important to the commercial grower. Nematodes are pandemic and their distribution extends nearly from pole to pole. In addition to their widespread distribution, various species of nematodes are etiological agents with diverse host ranges, including most plants and animals. For example, nematodes cause diseases as diverse as a pinworm disease in humans (Enterobius vermicularis is the etiological agent) to a root-knot disease in tomatoes. Although there are more than fifty species of root knot nematodes, the three most important species infecting tomato are Meloidogyne arenaria, M. incognita and M. javanica. 
Root knot species of nematodes are named for the type of root structure their infection produces in the plant. Upon successful infection, the nematode produces elicitors that result in the plant thickening its roots, resulting in the production of 1 mm to 10 mm lumps or galls on the roots. These changes facilitate the transport of nutrients from the plant to the nematode. The infected and morphologically altered roots have a concomitantly diminished capacity to supply water and nutrients to the plant. Plants manifest this reduced capacity for nutrient uptake with a general reduction of vigor that can be observed above ground. Infected plants may also display more specific symptoms such as stunting, wilting and chlorosis. As the nematode population builds up during the growing season, wilting becomes more pronounced and fruit set can be affected.
Chemical control of root knot nematodes is effective, and the nematicide methyl bromide provides excellent control for all of the Meloidogyne species. For various reasons, including health concerns for pesticide applicators and concern over ozone-depletion, the use of methyl bromide is being reduced around the world. An eventual ban of this chemical control agent highlights the importance of developing alternative methods to control nematode disease in tomatoes.
TYLCV is a geminivirus, and is classified in the Begomovirus group. Unlike the nematodes, which are ubiquitous in nearly all soil, the distribution of tomato yellow leaf curl virus is limited by the range of the insect vector that transmits the virus, the whitefly Bemisia tabaci. 
Commercial tomato production has shifted over the past twenty five years from temperate to more tropical growing regions in the world, due to lower labor costs, better transportation and treaties like the North American Free Trade Agreement and the World Trade Organization Agreement. This shift in geography coincides with the distribution of B. tabaci. Thus, as tomato growing regions have shifted towards tropical climates, the disease caused by TYLCV has become more pronounced. TYLCV can be rapidly transmitted to tomatoes by the feeding of the whitefly. Once in the plant, the virus replicates and spreads throughout the plant, although it is typically limited to the phloem. TYLCV causes severe symptoms in the plant, ranging from leaf curling and yellowing, to a stunting caused by a shortening of the internode length and arrest in floral growth. Together, this results in plants with a bush-like appearance. Crop losses can be severe; during the early 1990's, approximately 95% of the tomato crop in the Dominican Republic was lost, and in a single season (1991-1992), an approximate 140 million dollar loss was reported in Florida (Moffat, Science (1999) 286:1835).
Chemical control of the whitefly can be effective, but the misuse of pesticides in commercial tomato production has resulted in pesticide-resistant B. tabaci that can vector over 20 different tomato-infecting begomoviruses (Morales, (2001), Crop Protection; Poiston and Anderson (1997) Plant Disease 81:1358-1369; Zeidan et al. (1999) Asia Trop. Res. Ext. 1:107-115).
Just as the leading chemical option for nematode control in tomatoes is being phased out, chemical control options for TYLCV are also being reduced, for reasons ranging from the insect vector becoming resistant to the pesticide to public health concerns over the use of pesticides. The high development costs attendant to producing transgenic resistant cultivars is likewise an impediment to the development of TYLCV resistant cultivars using a genetic engineering approach. Those skilled in the art will thus recognize the need for alternatives to these chemical control strategies and transgenic strategies for the control of nematode, geminivirus (TYLCV) and other plant diseases in tomato production. The introgression of naturally occurring resistance genes remains the most effective option for controlling tomato pathogens today.
Although the inheritance of a resistance phenotype can be quantitative and polygenic, it is common in plants to have many dominant to semi-dominant resistance genes controlled by individual single loci. Plant resistance genes often encode for proteins that act as receptors that bind specific pathogen-encoded ligands. This pathogen-specific recognition and subsequent response by the plant is a phenomena first described by Flor in the late 1940's and referred to as ‘gene-for-gene’ resistance, (reviewed by Flor (1971) Ann. Review of Phytopathology 9:275-296). This specific receptor-ligand complex triggers a signal transduction pathway that ultimately results in a resistance phenotype (Baker et al. (1997), Science 276:726-733; Staskawicz et al. (1995) Science 268:661-667). In response to this recognition of pathogen attack, the host can respond with a strengthening of the cell wall, an oxidative burst, induction of defense gene expression and at times, rapid cell death at the infection site called the hypersensitive response.
For most breeding objectives, commercial breeders work with germplasm often referred to as the ‘cultivated type’. This germplasm is easier to breed with because it generally performs well when evaluated for horticultural performance. The performance advantage the cultivated types provide is often offset by a lack of allelic diversity. This is the trade off a breeder accepts when working with cultivated germplasm—better overall performance, but a lack of allelic diversity. Breeders generally accept this trade off because progress is faster when working with cultivated material than when breeding with genetically diverse sources.
In contrast, when a breeder makes either wide intra-specific crosses or interspecific crosses, a converse trade-off occurs. In these examples, a breeder typically crosses cultivated germplasm with a non-cultivated type. In such crosses, the breeder can gain access to novel alleles from the non-cultivated type, but has to overcome the genetic drag associated with the donor parent. Besides this difficulty with this breeding strategy, this approach often fails because of fertility or fecundity problems.
There are many wild relatives that can be crossed with cultivated tomato, including L. pennellii, L. hirsutum, L. peruvianum, L. chilense, L. parviflorum, L. chmielewskii, L. cheesmanii, L. cerasiforme, and L. pimpinellifolium. The genetic distance between the wild species and the cultivated L. esculentum correlates with the difficulty of both making the interspecific cross, and successfully creating a new commercial cultivar with an added trait (Genetics and breeding. MA Stevens and CM Rick. In: The tomato crop: A scientific basis for improvement. J G Atherton and J Rudich, editors. Chapman and Hall, (1994), London). For example, species like L. pimpinellifolium, L. cerasiforme, L. cheesmanii, L. chmielewskii and L. parviflorum are the easiest wild species to use as donors for trait introgression into the modern tomato. In contrast, L. pennellii, L. chilense, L. hirsutum and L. peruvianum are far more difficult species for trait introgression into the modern tomato (ibidem). When using these more distantly related species, it is not uncommon to have to use bridging species and embryo rescue for early generation crosses. Even with these extra steps, one can face significant segregation distortion, fertility problems, reduced recombination and genetic drag. Even in advanced generations, a suppression of recombination in the introgressed area of the genome presents the primary obstacle to reducing the genetic drag enough to create a successful commercial cultivar.
Thus, even though one may identify a useful trait in a wild species and target that trait for introgression into the cultivated species, there is no guarantee of success. Most successful commercial tomato breeders work their entire careers without successfully completing an introgression from a wild species to create commercial cultivars. The barriers to success include segregation distortion, which may result in areas of the wild genome that can be difficult to impossible to introgress. Further, some of the wild species of the modern tomato are self-incompatable, meaning that they cannot self pollinate. As a result of the self-incompatability, these plants are highly heterogeneous, having different alleles at many loci. The highly heterogenous nature of these wild species can also hinder the introgression of the most efficacious allele of interest.
The difficulty with introgressing novel alleles from wild relatives of domesticated crops extends to many crops, and is exemplified in tomato by a nematode resistance introgression. Bailey ((1941) Proc. Am. Hort. Sci. 38:573-575) first identified the wild species L. peruvianum as a potential source for nematode resistance. Smith ((1944) Proc. Am. Soc. Hort. Sci. 44:413-416) later used embryo rescue to successfully recover an interspecific hybrid containing the nematode resistance trait. Gilbert and McGuire ((1955) coined this locus Mi, and subsequently mapped Mi to chromosome 6 (Gilbert (1958) Tomato Genet. Coop Rep. 8:15-17). The resistance allele at the Mi locus, derived from L. peruvianum, is called the Mi-1 allele. The susceptible allele from L. esculentum, is referred to as the wild type allele, and designated ‘+’. It is believed that all commercial tomato cultivars containing the Mi-1 resistance allele are derived from the interspecific hybrid created by Smith. Although homozygous Mi-1 lines were developed as early as 1949 (Frazier and Dennett, (1949) Proc. Am. Soc. Hort. Sci. 54:225-236), it was not until the mid-1970's that the Mi-1 allele began to be commonly used in commercial cultivars. Two developments led to this commercial implementation. First, Rick and Fobes reported a linkage between an isozyme marker called alkaline phosphatase (Aps) and the Mi locus ((1974) Tomato Genet Coop. Rep. 24:25). The early use of a molecular marker test allowed breeders to follow the trait without performing pathology testing. Second, hybrid tomato cultivars were becoming more accepted by commercial growers. Despite breeding with the Mi-1 allele for six decades, there remains today significant genetic drag associated with the Mi-1 introgression from L. peruvianum, including a localized necrotic response (Ho et al. (1992) The Plant Journal, 2:971), smaller fruits and less fruit set under stress conditions.
Breeders found that the Mi-1 allele was efficacious when present as a heterozygote, which allowed them to deliver the nematode resistance trait to the hybrid from only one parent. This in turn allowed breeders to largely overcome the genetic drag by using a second inbred parent that have esculentum genes in this region of chromosome 6. The creation of hybrid cultivars allowed for the implementation of this breeding strategy. The dominant Mi-1 allele was bred into one of the inbred parents, and the susceptible allele (‘+’) with surrounding esculentum alleles that allowed for masking most of the genetic drag were bred into the second inbred parent. That the genetic drag could be masked in the heterozygous condition provides indirect evidence that there are genes in this region of the genome that affect the general health of the plant, fruit size and fruit yield because of the ability to respectively mask the localized necrotic response, smaller fruit size and less fruit set under stress conditions.
Plant resistance genes have been shown to be clustered in the genome, and in tomato, are commonly found near the centromeres. The Mi locus is located in one of these disease resistance clusters, near the centromere on chromosome 6. In addition to having the Mi resistance locus, other resistance genes for geminiviruses, Oidium lycopersicum (van de Beek et al. (1994) Theoret. Appl. Genet. 89:467-473), and two resistance genes for Cladosporium fulvum races 2 and 5 (Dickinson et al. Mol. Plant Microbe Interact. (1993) 6:341-347) are all tightly linked genetically in this centromeric region of chromosome 6.
The difficulty of the Mi-1 introgression, even after many decades, has been the inability to reduce the genetic drag associated with the trait. Alternate explanations for this difficulty are that the Mi-1 resistance gene is pleiotropic, and contributes to the genetic drag directly, or that there is a suppression of recombination in this genomic region that limits the progress of genetic drag reduction. Various experimental approaches have addressed this question. Using a combination of genetics and cytogenetics, Zhong et al. ((1999) Theoret. Appl. Genet. 98:365-370) showed that, based on the genome size of tomato, the physical distance between the Mi locus and the Aps locus should be about 750,000 base pairs, based on the genetic estimation of ˜1 cM of genetic distance. Their fluorescence in situ hybridization (FISH) results, however, showed that this physical distance is actually 40,000,000 base pairs. This discrepancy between the genetic and physical distances between these loci led Zhong et al. to predict that recombination around the Mi locus is reduced approximately 50-fold compared with the average for the genome. Kaloshian et al. ((1998) Mol. Gen. Genet. 257:376-385) took a comparative genetic approach, and showed that a L. peruvianum x L. peruvianum cross had 8-fold higher recombination in this region compared to the L. esculentum x L. peruvianum derived population. In addition to these experiments, it is well known that recombination is generally suppressed in centromeric regions. Milligan et al. (1998, Plant Cell 10: 1307-1320) used transgenic complementation to introduce the cloned Mi resistance gene into the susceptible cultivar Moneymaker. That no pleiotropic effects were observed in these complementation tests strongly suggests that the horticultural defects associated with the Mi introgression are due to genetic drag. These studies provided insight into the difficulty associated with introgressing disease resistance genes in this region of chromosome 6.
In 1998, Kaloshian et al. described a co-dominant, PCR-based molecular marker called REX-1, which was closer to the Mi locus than the Aps isozyme marker (Mol Gen Genet. 257:376-385). This DNA-based marker was rapidly adopted by tomato breeding programs, and greatly facilitated the development of new nematode resistance hybrid cultivars.
Although the tomato breeding community has rapidly disclosed its progress in introgressing the Mi-1 nematode resistance allele through scientific publications, the unlikelihood of success for this difficult breeding approach has been recognized by the USPTO in the issuance of several patents in this area (U.S. Pat. Nos. 6,414,226, 6,096,944, 5,866,764, and 6,639,132).
Reports of resistance to tomato yellow leaf curl geminivirus (TYLCV) have existed for nearly 40 years. Cohen first reported some tolerant genotypes as early as 1964 (Cohen and Harpaz (1964) Entomol. Exp. Appl. 7:155-166), then identified L. pimpinellifolium and L. peruvianum as containing higher levels of TYLCV resistance (Cohen and Nitzany (1966) Phytopathology 56:1127-1131). In the 1990's, Pilowski and Cohen reported tolerance from L. peruvianum (PI126935) with as many as five recessive genes (Plant Disease 74:248-250). Michelson et al. discovered ((1994) Phytopathology 84:928-933), and Hoogstraten (U.S. Pat. No. 6,414,226) later independently confirmed TYLCV resistance in L. chilense. This resistance locus is referred to as the Ty locus, and the resistance allele from L. chilense has been named Ty-1.
Like the Mi locus, the susceptible allele at the Ty locus is referred to as the wildtype, or ‘+’. Zamir et al. mapped the Ty locus to the centromeric region of chromosome 6 ((1994) Theoret. Appl. Genet. 88:141-146). The Ty-1 allele acts as a dominant allele, thus both lines that are fixed for the Ty-1 allele or that are heterozygous (Ty-1/‘+’) are resistant to TYLCV.
The inbred tomato line FDR16-2045, containing the Ty-1 resistance gene from L. chilense also confers resistance to nematodes because of a resistance gene from L. chilense that was also introgressed at the nearby Mi locus (Hoogstraten, U.S. Pat. No. 6,414,226). That these two resistance genes for nematodes and geminiviruses are co-inherited in line FDR16-2045 demonstrates that the Ty and Mi loci are closely positioned genetically. The nematode resistance allele at the Mi locus, as introgressed from L. chilense in line FDR16-2045 is referred to as Mi-J. Line FDR16-2045 is a valuable breeding inbred because it allows breeders to create commercial hybrids containing efficacious resistance alleles for nematodes and geminiviruses, with the ability to countermand most of the genetic drag by using a second inbred parent with the ‘+’ type alleles at the Mi and Ty loci. The genetic drag from this introgression can be manifested as autonecrosis, longer internodes, smaller fruits and less fruit set under stress conditions.
However, through pathology testing it has been found that the Mi-J allele from L. chilense is not as effective as the Mi-1 allele from L. peruvianum. This is particularly evident when the Mi-J allele is paired in an F1 hybrid with the ‘+’ susceptible allele at the Mi locus. Using molecular techniques, the present inventors were able to design molecular marker tests to distinguish the three possible alleles (Mi-1, Mi-J and ‘+’) at the Mi locus.
Tomato breeders are faced therefore with a limitation in their ability to deliver multiple resistance genes that map to the centromeric region of chromosome 6 while retaining the ability to mask the genetic drag associated with these introgressions. To pyramid all the known resistance genes that map in this region of chromosome 6 in a hybrid cultivar, a breeder would have to have one parent with the introgression from L. peruvianum containing the nematode resistant gene Mi-1, another parent with the introgression from L. chilense containing the TYLCV resistance gene Ty-1, another parent with the introgression from L. hirsutum containing the resistance gene for Oidium, another parent with the introgression from L. pimpinellifolium containing the resistance genes for races of Cladosporium, and yet another parent containing the ‘+’ type alleles from esculentum in order to mask the genetic drag associated with some of these introgressions. This task is impossible for the breeder because they have only two parent lines to choose from to make hybrid cultivars. This dilemma is also shown graphically by Ho et al. ((1992) The Plant Journal 2:971-982, see FIG. 6), and by Liharska et al. ((1996) Genome 39:485-491, see FIG. 1).
Thus, there remains a need to identify a recombinational event in this area of the genome known to have severely suppressed recombination, and that will contain the most efficacious allele for nematode resistance, Mi-1, originally introgressed from L. peruvianum, with the most efficacious allele for TYLCV resistance, Ty-1, originally introgressed from L. chilense. Tightly linked alleles juxtapositioned in this manner are said to be in the coupling phase, or in cis. Such a combination of efficacious resistance alleles in cis would allow tomato breeders to create tomato hybrids with the most efficacious resistance to TYLCV and nematodes, while retaining the freedom of having a second inbred parent to either mask the genetic drag, or deliver additional resistance genes, such as the resistance genes for Oidium, Cladosporium, or yet to be discovered resistance alleles in this disease cluster.